Serum calprotectin as a marker of neonatal sepsis: a hospital-based cross-sectional diagnostic study [version 1; peer review: 2 approved]

Background: Despite significant advances in neonatal care, neonatal sepsis remains a major contributor to mortality, morbidity


Introduction
Sepsis is a severe and potentially lethal organ dysfunction often produced by an inadequate host response to an infection. 1 Neonates are a unique cohort of populations showing differences in physiology and immunology between children and adults. Even though the last decade has shown a substantial reduction in neonatal mortality globally, septicemia continues to be a significant contributor, accounting for 11 million neonatal deaths yearly. 2 In neonates the onset of sepsis is often quiet with minimal unclear and nonspecific signs. An accurate and early diagnosis plays a critical role. It is for this purpose that a host of novel biomarkers are being explored. The most significant aspect is that they are markers of adaptive immunological responses that are not well established during the initial post-natal period. The gold standard for organism isolation remains to be blood culture. Nevertheless, the culture results are only available after 48 hours and, sometimes, fail to show the growth of microbes despite the clear clinical picture of sepsis. Hence, neonatal sepsis remains challenging for clinicians to ensure accurate diagnosis at the appropriate time.
Calprotectin, also known as MRP 8/14, S100A8/S100A9, is a zinc and calcium-binding protein heterodimer. It is primarily located in the cytosolic neutrophil fraction and comprises nearly 30-40% of the protein content. 3 It is released into the circulation due to exocytosis of granules from activated neutrophils. 4 Calprotectin intracellular roles include activation of neutrophilic NADPH oxidase and cytoskeletal regulation of phagocyte migration. 5,6 It contains apoptosisinducing, antibacterial, proinflammatory, and oxidant-scavenging properties. 7,8 In vitro studies have demonstrated bacteriostatic, fungi-static, and resistance to enzymatic degradation. Calprotectin elevation in extracellular fluids of inflammatory disorders such as abscesses, cystic fibrosis, and rheumatoid arthritis have been reported. [9][10][11][12] Calprotectin is secreted into circulation by innate immune system cells immediately following a host-pathogen interaction. An enzyme-linked immunosorbent assay (ELISA) test can detect it. Its potential use in the diagnosis of various inflammatory diseases is being investigated. Earlier studies have shown different cut-off levels, varying sensitivity, and specificity of serum calprotectin in detecting sepsis in neonates. [13][14][15][16] The literature on its diagnostic value in newborn sepsis in the Indian context is, however, scarce.
Procalcitonin is one of the most widely studied markers in sepsis. Monocytes and hepatocytes produce procalcitonin, which rises within four hours and has a half-life between 25 and 30 hours. 17 It is thus a reliable indicator of sepsis compared to conventional markers.
According to a comprehensive review and meta-analysis by Vouloumanou et al., procalcitonin has a pooled sensitivity and specificity of 81% and 79% in identifying newborn sepsis. 18 According to a recent meta-analysis by Ruan et al., procalcitonin paired with C-reactive protein (CRP) or presepsin was more accurate in diagnosing newborn sepsis. They also stated the various cut-off levels used to define neonatal sepsis ranged from 0.5 to 2.2 ng/ml. 19 Procalcitonin levels increase physiologically in the first 24 hours of life, with elevations additionally seen in non-infectious causes such as trauma, surgery, and respiratory distress syndrome. These confounding factors limit the utility of procalcitonin in neonatal sepsis, specifically in early-onset sepsis. 15 This study aimed to estimate calprotectin levels in newborns with clinical sepsis and compare its diagnostic accuracy with other sepsis markers such as blood culture, CRP, and serum procalcitonin.

Study setting
We conducted cross-sectional diagnostic research at Tertiary Neonatal Critical Care Units linked to Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal, India. We included admitted neonates with clinical sepsis diagnosed by clinical criteria 20 between December 2018 to September 2020 by convenient sampling.

Study design
This research follows the Standards for Reporting Diagnostic Accuracy (STARD) 21 statement guidelines. The reporting guidelines contain a completed STARD checklist. 22 Figure 1 depicts the study flow according to STARD criteria. 22 Sample size Based on a prior study by Terrin et al., 13 where the sensitivity of serum calprotectin levels in predicting neonatal sepsis was 89% and using a normogram with 10% absolute precision, 95% confidence interval, with 10% non-responsive rate, the sample size was estimated to be 77 and rounded off to 80.

Ethics and data collection
The 1964 Declaration of Helsinki and its later amendments, as well as other related ethical principles, were followed in the conduct of the study. The Institutional ethics committee of Kasturba Medical College, Mangalore (IEC KMC MLR 10-18/378, dated 17/10/2018) approved the study, and we obtained appropriate hospital authorities' permits. We approached the parents and guardians of newborns who met the inclusion criteria and informed them in their native tongue about the study's goals. We gave the parents a participant information sheet with answers to the most frequently asked queries (as in Extended data). 22 We obtained signed informed consent (as in Extended data) 22 if the parents or guardians were willing for their newborns to participate. We gathered the baseline demographic information and neonatal medical history using a validated semi-structured pretested proforma (as in Extended data). 22 Inclusion and exclusion criteria Admitted neonates with clinical sepsis diagnosed as per the clinical criteria were included. We excluded ventilated neonates with respiratory or circulatory failure, previous exposure to antibiotics, preterm less than 32 weeks, had APGAR scores less than "3" or had conditions such as persistent pulmonary hypertension of newborn, congenital malformations, surgical-related disorders, and severe intracranial bleeding.

Operational definitions
Clinical sepsis in newborns was defined as the presence of two or more of the following characteristics, namely respiratory instability, cardiovascular instability, gastrointestinal instability, temperature instability, sclerema or petechial rash, and nonspecific features. 20 We considered sepsis screen to be positive if two or more of the following observed laboratory parameters, namely CRP >6 mg/dl, total leucocyte count (TLC) >20,000Â10 9 /L or <4,000Â10 9 /L and immature to total neutrophil ratio (I/T ratio) >0.2. 20 The sex of the neonate as male or female was determined by external examination of body characteristics by the investigator.

Sample collection
We collected a venous blood sample to estimate TLC, I/T ratio, CRP, blood culture, serum procalcitonin, and serum calprotectin levels. Beckman Coulter's automated system and Nephelometry calculated the total leucocyte count and CRP, respectively. A peripheral smear examination differentiated the leucocytes, and we determined the I/T ratio. For blood cultures, we collected about 1 mL of venous blood under aseptic conditions. We inoculated blood into blood culture medium bottles (BD BACTEC™ PedsPlus™/F culture vials) before being evaluated for growth at regular intervals.
For serum procalcitonin and serum calprotectin levels estimation, we collected an aliquot of 2 ml venous blood in plain tubes, centrifuged it at 5,000 rotations per minute for 15 minutes, and kept the separated serum at -80°C until further processing. Serum calprotectin levels were estimated using the Human CALP (Calprotectin) ELISA Kit (Catalog no: ELK4602) from ELK Biotechnology ® , China, on LISA plus ELISA reader. The detection range for the calprotectin kit was between 31.25 and 2,000 pg/ml, and its sensitivity was 13.7 pg/ml with high specificity. Serum procalcitonin was estimated using the Human PCT (Procalcitonin) ELISA Kit (Catalog No: E-EL-H1492) from Elabscience ® , China, on LISA plus ELISA reader. The procalcitonin kit exhibited a sensitivity of 18.75 pg/ml, a detection range of 31.25 to 2,000 pg/ml, and a coefficient of variation under 10%. We converted the measured values of serum calprotectin and procalcitonin into ng/ml since the conventional expression of serum procalcitonin is in ng/ml.

Statistical analysis
We used IBM SPSS Statistics (RRID:SCR_016479) for Windows, Version 25.0 (IBM Corp., Armonk, NY) to analyze the data. We expressed patient characteristics in proportions. We calculated the median and interquartile ranges for the following parameters: TLC, CRP, I/T ratio, serum calprotectin levels, and procalcitonin levels. By using Mann-Whitney U test, we compared the values of serum calprotectin and other conventional markers of sepsis among culture-positive and culture-negative sepsis. We performed a receiver operator characteristic (ROC) curve to obtain a cut-off for serum calprotectin levels to detect sepsis in neonates when compared with the conventional sepsis parameters/serum procalcitonin.

Results
Of the 83 neonates included, 63% (52/83) were male, 79.5% (66/83) were inborn babies, and 54% (45/83) were delivered by cesarean section. The median gestational age and birth weight values were 261 (IQR: 244-274) days and 2,100 (IQR: 1,600-2,800) grams, respectively. The median length of stay in the NICU was 6 (IQR: 5-11) days. The median values of CRP, TLC, serum calprotectin, and serum procalcitonin are presented in Table 1. 22 There was no significant association  Klebsiella and Methicillin-Resistant Staphylococcus aureus (MRSA) were isolated in six cases each, followed by Acinetobacter in four patients. Further, we documented Citrobacter and Pseudomonas growth in three cases each and Methicillin-sensitive Staphylococcus aureus in one case. Table 2 compares the median serum calprotectin, procalcitonin levels, and conventional markers of sepsis among the blood culture-positive and culture-negative cases. The differences in the median values of serum calprotectin, procalcitonin, TLC, and I/T ratio between blood culture-positive and culture-negative groups were not statistically significant.
The area under the curve (AUC) was 0.39 (S.E. 0.06, p=0.1, CI=0.27 to 0.51) when serum calprotectin was compared to blood culture using a ROC curve (Figure 3). We determined the sensitivity and specificity to be 50% and 44%, respectively, at a cut-off level of 0.83 ng/ml. The serum calprotectin ROC curve exhibited an AUC of 0.536 (S.E. 0.08, p=0.69, CI=0.37 to 0.70) compared to CRP (Figure 4). We discovered the sensitivity and specificity to be 57% and 67%, respectively, at a cut-off level of 0.89 ng/ml of calprotectin. We determined the sensitivity and specificity to be 60% and 73% at a 1.2 ng/ml cut-off level of serum calprotectin.

Discussion
Calprotectin is an innate immune marker; thus, we investigated its significance as a biological marker for the early diagnosis of newborn septicemia. We found the median serum calprotectin levels in clinically septic neonates to be 0.93 ng/ml (IQR 0.67-1.3). Earlier studies documented the median or mean serum calprotectin levels in septic, nonseptic, and control neonates. [13][14][15][16]23 The varied broad range of reported values may be because of the differences in the kit employed for estimation.
We observed that the serum calprotectin was higher in the blood culture positive group as compared to the culture negative group, however the difference in the current study was not statistically significant. Similar to this, previous studies found higher calprotectin levels in blood culture positive groups compared to negative groups, [14][15][16]23 with statistically significant differences. 13,23 In addition, in contrast with previous research, we were unable to determine the precise reason why our study cohort had lower median serum calprotectin levels. Possible explanations include earlier studies evaluated serum calprotectin in particular neonatal groups, namely very low birth weight 13 and late-onset sepsis. 23 The current study, however, comprised a broad sample of newborns with early and late-onset sepsis and a range of birth weights, offering a unique perspective that calls for further subgroup research. Furthermore, the kits used in various studies were different.
Respiratory instability was the most common manifestation seen in more than two thirds of cases (68%) in our study and was similar to the survey by Attia et al. 15 In the current study, calprotectin levels did not significantly differ between males and females, which is consistent with other research. 15,23 Blood culture-positive cases contributed to 36.5% of the neonates in our research and are in line with the expected culturepositivity rates of 10 to 30% in neonatal sepsis. 24 Previous studies have documented blood culture-positive cases in 19.5% 14 , 16.6% 23 and 75% 15 cases. The most common isolates in the present study were Klebsiella and MRSA and were concurrent with the published literature. 14,15 When compared with CRP, blood culture, and serum procalcitonin levels, the ROC curves generated sensitivity and specificity levels with cut-off values of serum calprotectin to detect neonatal sepsis. In the current study, serum calprotectin had a sensitivity of 60% and specificity of 73% compared to serum procalcitonin in identifying newborn sepsis at a 1.2 ng/ml cut-off. In the literature, limited data compare serum calprotectin with serum procalcitonin levels in neonates. However, our study showed poor sensitivity and specificity concerning the gold standard blood culture. The correlation of serum calprotectin with blood culture was not documented in previous studies. [13][14][15][16]23 Serum calprotectin had sensitivity between 42.5-92% and specificity between 70-96% with cut-off levels between 1.4 to 38.3 μg/ml, as per earlier studies. [13][14][15][16]23 The wide variations in the values may be due to different kits and a lack of international standards. It is unclear how our serum calprotectin levels were lower than that documented in previous studies. Possibilities of varying kit specifics and influences from the diverse neonatal population may be contributory, this being the first Indian neonatal population studied.
This study's limitations were the lack of a control group for comparison, the relatively smaller sample size, and the sensitivity and detection range of the ELISA kit used to estimate serum calprotectin. Further multicenter studies involving a larger population of neonates are warranted given broad ranges of mean/median values of serum calprotectin, varied ranges of sensitivity and specificity with different cut-off values, and due to varying usages of kits in various studies.

Conclusions
Serum calprotectin is not superior to existing sepsis markers. Serum calprotectin levels equal to and above 1.2 ng/ml had a sensitivity of 60% and specificity of 73% compared to serum procalcitonin in detecting sepsis in our neonatal population.

Is the study design appropriate and is the work technically sound? Yes
Are sufficient details of methods and analysis provided to allow replication by others? Yes

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Yes
Rathika Damodara Shenoy Department of Paediatric Surgery, K.S. Hegde Medical Academy, NITTE (Deemed to be University), Mangalore, Karnataka, India Newborn sepsis often presents with non-specific symptoms and signs. Blood cultures are positive in just about one-third. There is a constant search for a sensitive and specific biomarker, and this study has investigated the suitability of serum calprotectin. The comparison has been made against culture-positive sepsis, elevated CRP and procalcitonin. The methodology is well written. The cut-off of PCT used to determine ROC in the study can be added either under Operational definitions or under Statistical analysis sections.
The median birth weight of the study population of 83 newborns is 2100g. This suggests that the study population was comprised largely of a small gestation-age newborns. It is surprising to note